Hsp90 senses proteotoxic stress and provides obligatory support to a diverse family of normal and mutant signal transduction proteins. To fulfill these roles, Hsp90 associates with networks of non-client co-chaperone partners to generate an unknown number of discrete Hsp90 "machines." These networks appear to regulate or target Hsp90 function. Due to Hsp90's pivotal role in normal and aberrant cell physiology, Hsp90 inhibitors are in clinical trials to treat human disease, and the expanding network of known co-chaperones includes several proto-oncoproteins and several noteworthy pharmacological targets. Our long-term goal is to define the roles of Hsp90 and its co-chaperones, and to elucidate the mechanisms underlying their functions. The evolving field of proteomics now provides the tools needed to examine the composition, organization, and dynamic regulation of the Hsp90 co-chaperone network. Thus, this R21 application proposes to initiate the discovery novel Hsp90 co-chaperones, to confirm their interactions with Hsp90, and to determine if individual Hsp90-binding proteins behave as co-chaperones versus clients. Additionally, we propose to test the hypothesis that the Hsp90 co-chaperone network is regulated by proteotoxic cell stresses such as hyperthermia, heavy metal, and ethanol. These Aims will be pursued by isolating the Hsp90 co-chaperone network via one-step purification, separating and quantifying Hsp90-associated proteins via 1-D and 2-D electrophoresis, and by using digital imaging and data-basing software to compare Hsp90 co-chaperone networks isolated from stressed versus unstressed tissues. Individual Hsp90-associated proteins will be identified by trypsinolytic fingerprinting and MALDI-based mass spectrometry. Associations with Hsp90 will be confirmed via reciprocal immunoadsorptions. Authentic co-chaperones will be differentiated from Hsp90 clients on the basis of the prototypical effects of Hsp90 antagonists. Potential regulatory mechanisms will be examined via biochemical characterizations of dynamically responsive co-chaperones. The research proposed will elucidate the composition and dynamic regulation of a discrete protein network that supports human development, and will characterize how environmental stress may dysregulate this network.